Previously, we demonstrated the existence of a set of eight genes that are essential for the secretion of pertussis toxin (PT) from B. pertussis. These genes, termed ptl genes, encode transport proteins that share homology with a family of proteins involved in the transport of both DNA and proteins across bacterial membranes. We now have evidence for a ninth secretion gene. This small gene, ptl1 is predicted to encode a protein of 6.8 kDa and is located between ptlD and ptlE. PtlI shares homology with VirB7 which is required for transport of T-DNA across the membranes of the plant pathogen Agrobacterium tumefaciens. Deletion of the ptlI region from B. pertussis has no discernable effect of PtlE but resulted in loss of detectable PtlF, suggesting the PtlI may interact with and stabilize PtlF. Using immunoprecipitation techniques, we demonstrated a direct interaction between PtlI and PtlF. These results are the first evidence of interactions between Ptl proteins. In addition to the work on elucidation of interactions between Ptl proteins, we are also investigating the mechanism by which the Ptl proteins promote transport of PT. One Ptl protein in particular, PtlC, has the interesting characteristic that it contains a nucleotide binding motif. We are currently purifying this protein and determining whether this protein has either hydrolase or kinase activity that might play a critical role in the transport process. This work increases our knowledge of the secretion of PT and may aid in the construction of strains of B. pertussis which efficiently produce and secrete PT. Such strains would be useful for vaccine production. In addition, knowledge of the mechanism of PT secretion might aid in the development of live-attenuated vaccines that secrete inactivated forms of PT, thus inducing a protective immune response.